The current invention relates to the design, synthesis, and biochemical evaluation of chromogenic substrate compounds for sialidases of bacterial, viral, protozoa, and vertebrate (including humans) origin. In particular, this invention provides a novel class of effective compounds as chromogenic substrates of these sialidases which yield chromogenic products after reactions catalyzed by sialidase take place. Also provided are methods of making these substrate compounds, methods of diagnosis and prognosis of sialidase related diseases using these substrate compounds.
Sialidase (EC, 3.2..1.18, also known as neuraminidase, acylneuraminyl hydrolase) is a protein enzyme produced by many organisms such as bacteria, viruses, protozoa, and vertebrates including humans (Hirst, G. K. [1941] Science 94:22-23). This class of enzymes catalyzes the hydrolysis of a terminal sialic acid which is linked to oligosaccharides through an O-glycosidic bond. Crystal structure of sialidases showed that the enzyme has a highly conserved active site centered in a propeller like xcex2-sheet twirl (Crennell, S. J. et al. [1993] Proc. Natl. Acad. Sci. USA 90:9852-9856).
Sialidases perform many critical biological functions. In bacteria, sialidase helps bacterial adhesion to tissues, and provides additional nutritional sources (Crennell, S. et al. [1994] Structure 2(6):535-544). In viruses, it helps the release of progeny viruses (Liu, C. et al. [1995] J. Virol. 69:1099-1106). In a parasite, Trypanosoma cruzi, a sialidase (also known as trans-sialidase) removes sialic acids from infected cells and decorates its own surface with these sialic acids. In humans, sialidases are involved in protein digestion, immune responses, and cell proliferation. Abnormal production of sialidases may lead to serious human diseases such as sialidosis or increased Pseudomonas aeruginosa infection in cystic fibrosis patients.
Since sialidases are associated with many diseases, a color-producing substrate of sialidase would be an excellent diagnostic or prognostic reagent for sialidase-related diseases. For instance, sialidase level is elevated in bacterial vaginosis (Briselden, A. M. et al. [1992] J. Clin. Microbiol. 30:663-666). Measurement of sialidase level in the vaginal samples could be used to diagnose bacterial vaginosis. In periodontal disease caused by bacterial infection, it has been shown that presence of sialidase increases the colonization of harmful bacteria (Liljemark, W. F. et al. [1989] Caries Res. 23:141-145). The cell invasion form of T. cruzi, Trypomastigote, expresses high levels of trans-sialidase activity; therefore, measurement of trans-sialidase level could be used for diagnosis of T. cruzi infection and for monitoring disease progress (Cross, G. A., G. B. Takle [1993] Annu. Rev. Microbiol. 47:385-411). In cystic fibrosis patients, Pseudomonas aeruginosa infection is one of the leading causes of death. Sialidase was shown to be involved in the disease progress (Cacalano, G. et al. [1992] J. Clin. Invest. 89:1866-1874). Sialidase is also related to the regulation of cell proliferation (Bratosin, D. et al. [1995] Glycoconj. J. 12:258-267), the clearance of plasma proteins (Bonten, E. et al. [1996] Genes and Devel. 10:3156-3169), and the catabolism of gangliosides and glycoproteins (Gornati, R. et al. [1997] Mol. Cell Biochem. 166:117-124).
Currently, there is available a synthetic substrate of sialidase, 4-methylumbelliferyl-B-acetyl-neuraminic acid (4-MUN) (Lentz, M. R., R. G. Webster, G. M. Air [1987] Biochemistry 26:5351-5358), which produces a product with characteristic fluorescence spectrum upon hydrolysis. This change of fluorescence spectrum can only be measured with a specialized instrument (fluorospectrometer). The substrate compounds of the current invention produce a visible color change upon hydrolysis, which is highly advantageous in medical diagnostic applications.
In one embodiment, the current invention relates to the design and synthesis of novel chromogenic substrate compounds for sialidases. In another embodiment, the subject invention pertains to the use of the novel chromogenic substrates in assays for the detection of sialidases. The sialidases which are detected using the procedures and compounds of the subject invention are of bacterial, viral, protozoa, and vertebrate (including human) origin. In a specific embodiment, the subject invention provides a novel class of compounds which are useful as chromogenic substrates of sialidases.
In one embodiment, the present invention provides chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, NHC(O)R6, NHC(O)OR6, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein R2=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, NHC(O)R6, NHC(O)OR6, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein R4=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, NHC(O)R6, NHC(O)OR6, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2,)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein R5=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, NHC(O)R6, NHC(O)OR6, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2,)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R3=NO2, CHO, (CR8xe2x95x90CR8)kCN or (CR8xe2x95x90CR8)kNO2, where k is an integer from 1 to 3, or 
wherein, R6=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R7=R6, OR6, or N(R6)2; wherein R8=H or (CH2)nCH3; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds having the formula of General Structure I, wherein, R1=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R2 or R4=H, R6, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R3=H, R6, OR6OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R5=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R2 or R4=NO2, CHO, (CR8xe2x95x90CR8)kCN or (CR8xe2x95x90CR8)kNO2, where k is an integer from 1 to 3, or 
wherein, R6=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R7=R6, OR6, or N(R6) 2; wherein R8=H or (CH2)nCH3; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds having the formnula of General Structure I, wherein, R1 or R5=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R2=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R3=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2),CH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R4=H, OR6, OC(O)R7, NO2, NH2, N(R6)2,Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein R1 or R5=NO2, CHO, (CR8xe2x95x90CR8)kCN or (CR8xe2x95x90CR8)kNO2, where k is an integer from 1 to 3; wherein, R6=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R7=R6, OR6, or N(R6)2; wherein R8=H or (CH2)n CH3; where n is an integer from 0 to 3.
Also provided are chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R2=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R3=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R4=H, OR6, OC(O)R7, NO2, NH2, N(R6)2, Cl, Br, I, F, CHO, CO2R6, C(O)N(R6)2, C(Nxcx9cOH)NH2, OPO3R6, OPO2(CH2,)jCH3, CH2PO3R6, OSO3R6, OSO2(CH2)jCH3, CH2SO3R6, or CN, where j is an integer from 0 to 3; wherein, R5=H or (CH2)kCH3, where k is an integer from 0 to 4; wherein, R6=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R7=R6, OR6, or N(R6)2.
Also provided are chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, OR3, OC(O)R4, NO2, NH2, N(R3)2, Cl, Br, I, F, CHO, CO2R3, C(O)N(R3)2, C(Nxcx9cOH)NH2, OPO3R3, OPO2(CH2)jCH3, CH2PO3R3, OSO3R3, OSO2(CH2)jCH3, CH2SO3R3, or CN, where j is an integer from 0 to 3; wherein, R2=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R3=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R4=R3, OR3, or N(R3)2.
Also provided are chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, OR3, OC(O)R4, NO2, NH2, N(R3)2, Cl, Br, I, F, CHO, CO2R3, C(O)N(R3)2, C(Nxcx9cOH)NH2, OPO3R3, OPO2(CH2)jCH3, CH2PO3R3, OSO3R3, OSO2(CH2)jCH3, CH2SO3R3, or CN, where j is an integer from 0 to 3; wherein, R2=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein R3=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R4=R3, OR3, or N(R3)2.
Also provided are chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R2=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R3=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R4=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R5=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R6=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R7=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)CH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein R8=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R9=R8, OR8, or N(R8)2.
Also provided are chromogenic sialidase substrate compounds having the following formula: 
wherein, R1=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2),CH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R2=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R3=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R4=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R5=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R6=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R7=H, OR8, OC(O)R9, NO2, NH2, N(R8)2, Cl, Br, I, F, CHO, CO2R8, C(O)N(R8)2, C(Nxcx9cOH)NH2, OPO3R8, OPO2(CH2)jCH3, CH2PO3R8, OSO3R8, OSO2(CH2)jCH3, CH2SO3R8, or CN, where j is an integer from 0 to 3; wherein, R8=H, C(CH3)3, CH(CH3)2, CH2CH(CH3)2, CH(CH3)(CH2)mCH3, or (CH2)mCH3, where m is an integer from 0 to 3; wherein, R9=R8, OR8, or N(R8)2.
The subject invention further pertains to analogs, salts, derivatives, and mixtures of the subject compounds.
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